! PROJECT SUMMARY Precise regulation of gene expression is critical for development and cell identity. Misregulation of this tightly controlled process can lead to disease such as cancer or neurological disorders. Distinct epigenetic marks (DNA methylation and post-translational histone modifications) have been associated with expressed or silenced genes as well as with regulatory elements in the genome. Traditionally, drugs (e.g., decitabine, vorinostat) are used to induce epigenetic changes in an untargeted manner and thus can alter epigenome signatures throughout the genome. With the RNA-guided Cas9/CRISPR complex, we now have a tool that can easily and precisely target a 20-bp sequence in the genome. We and others have developed targeted epigenetic regulators that are based on fusions of effector domains to the catalytically inactive dCas9. We have shown that dCas9 fused to various epigenetic effector domains (epi-dCas9) can regulate transcription in a targeted manner. However, two major challenges have to be overcome before we can use these tools efficiently: 1) Efficiency of epigenetic regulators is dependent on the genomic locations. Pre-existing chromatin environment and three- dimensional interactions that make a target locus amenable to persistent targeted epigenome editing are not yet understood. 2) The factors and pathways to efficiently achieve persistent targeted gene silencing are not well defined. Clearly, a better understanding is needed of the targetable epigenome that is amenable to persistent gene silencing and an understanding of the pathway(s) to accommodate persistent gene silencing. We will gain these foundational insights by determining promoter features amenable to persistent targeted gene silencing by epi-dCas9 (Aim 1), and identifying pathway(s) required for persistent target gene silencing using an innovative epi-dCas9/knockdown editing screening system (Aim 2). In the first Aim, we will test several epi-dCas9 fusions on 80 promoters representing different expression levels and epigenetic states, then identify features that are permissive or resistive to persistent silencing. In a parallel Aim, we will combine an epi-dCas9 repressor with a genome- wide CRISPR/Cas9 screen to identify cellular genes involved in epigenetic persistence. Not only will this information advance the capabilities of us and others to create targeted persistent epigenetic changes for the study and treatment of disease, it will also provide fundamental insights into the mechanistic steps required to transition from one epigenetic state to another. !